长链非编码RNA-XIST在肝癌转移相关性氨基酸代谢重编程中作用的研究

Hepatocellular carcinoma，which grows rapidly and tends to metastasize with serious adverse prognosis, is one of the most common malignant tumors. The current mechanism of hepatocellular carcinoma growth and metastasis is still unclear. Cancer metabolic reprogramming has been recognized as one of the ten cancer hallmarks. Cancer cell favors the shift to glycolytic metabolism, referred to as "the Warburg effect," a phenomenon whereby cancer cells rely mainly on aerobic glycolysis to generate ATP even in the presence of O2. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically amino acid metabolism which is highly transported into proliferating cells, is a major source for energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of amino acid metabolism in hepatocellular carcinogenesis is not well understood. Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides with limited protein coding potential. Recently, many studies have shown that lncRNAs are frequently deregulated in various diseases and have multiple functions in a wide range of biological processes, such as proliferation, apoptosis, or cell migration. However, the specific roles of lncRNAs in mediating the function in regulating Warburg effect of HCC is still not well studied. To determine the overall impact of lncRNAs on hepatocellular carcinogenesis, we analyzed the expression profile of lncRNAs in 10 pairs of human HCC samples using microarray analysis. Our results demonstrated that LncRNAs：CRNDE、SNHG1、ZFAS1、XIST、C11orf95、SNHG16 and RP11-151N17.2 have statisticly aberrant expression in HCC of high metastatic potential compared with non-tumor liver tissue and are relatively correlated with survival of HCC patients. More importantly, a positive correlation was observed between the expression of XIST and Glutaminase 2 (GLS2), an enzyme that converts glutamine to glutamate. Our preliminary data suggested that GLS2 can significantly inhibit the proliferation of HCC through the regulation of PI3K/AKT pathway. According to the above, the current project proposes the supposition that LncRNA XIST would be able to regulate amino acid metabolism mediated HCC metastasis. To verify this hypothesis, we will perform experiments using the methods of RIP, Luciferase, orthotopic xenograft nude mice model and metastasis model, PET scan to investigate the influences of LncRNA XIST. The project will provide new therapeutic targets and interventions for HCC from the perspective of lncNA XIST and amino acid metabolism.

肝癌是消化系统最常见恶性肿瘤之一，其生长快速且易发生转移，多预后不良，目前肝癌生长及转移机制尚不清楚。“Warburg效应”是肿瘤细胞优先以糖酵解作为提供能量的主要方式，近年发现肿瘤细胞不仅糖代谢异常，氨基酸代谢也有类似变化，而氨基酸代谢对肝癌生长转移的影响尚无研究。研究表明长链非编码RNA（lncRNA）参与包括肿瘤生长在内多种疾病过程调控。我们前期利用lncRNA芯片筛选，分析出与肝癌预后相关的15个lncRNA，并结合与氨基酸代谢调控相关的主要关键酶，最终筛选出了LncRNA XIST，由此我们猜想，XIST是否通过调控氨基酸代谢从而调控肝癌的生长及转移。本课题拟以XIST为研究对象，利用RNA免疫共沉淀、裸鼠原位肝癌模型的小动物活体成像及PET-CT等方法，从多层面研究XIST对氨基酸代谢的影响进而调控肝癌的生长及转移，率先从氨基酸代谢角度为肝细胞肝癌的治疗提供新思路与借鉴。

项目背景：.肝细胞肝癌是全球第五大常见肿瘤。根据欧洲癌症协会统计数据显示，肝细胞肝癌占所有原发性肝癌的 80％以上，全世界每年约有50至 100 万人死于肝细胞肝癌，90％癌症患者死于肿瘤转移造成的并发症，而非原发肿瘤，肝细胞肝癌是一种高度富含血管且常发生肝内外转移的恶性肿瘤，肝细胞肝癌患者术后复发快，且预后不良，多数肝细胞肝癌患者死于癌症局部复发或术后短时间内发生的转移性疾病。然而，肝细胞癌发生发展的机制仍不清楚。近年发现肿瘤细胞不仅糖代谢异常，氨基酸代谢也有类似变化。LncRNA XIST是氨基酸代谢调控相关的主要关键酶，但其对肝癌生长转移的影响尚不明确，因此本文主要探索LncRNA XIST在肝癌转移相关性氨基酸代谢重编程中的作用。.主要研究内容：.检测LncRNA XIST在肝癌患者肿瘤组织中的表达情况，并结合肝癌患者的临床病理学资料分析LncRNA XIST表达与肝癌患者预后的关系；在体内外对肝细胞癌增殖、转移及warburg效应的影响。应用克隆形成实验、细胞增殖实验、细胞周期和凋亡评估和裸鼠体外移植瘤实验检测LncRNA XIST在肝癌细胞增殖、凋亡及转移的影响。应用RNA免疫共沉淀、裸鼠原位肝癌模型的小动物活体成像及PET-CT等方法，从多层面研究XIST对氨基酸代谢的影响进而调控肝癌的生长及转移，从氨基酸代谢角度为肝细胞肝癌的治疗提供新思路与借鉴。.重要结果及关键数据：.对肝癌细胞系中沉默或过表达分析LncRNA XIST后观察肝癌细胞的增殖、凋亡、细胞周期及转移变化。同时过表达或沉默LncRNA XIST，我们已明确LncRNA XIST在体内外促进肝细胞肝癌增殖和转移中起到重要作用，可促进肝细胞肝癌的warburg效应。lncRNA XIST可通过GLS2抑制肝癌细胞的谷氨酰胺代谢进而促进肿瘤的进展。初步机制研究发现LncRNA XIST可以与 miR-424竞争性结合，以增加其对靶基因GLS2的促进作用，发挥促癌作用。LncRNA XIST/miR-424 /GLS2途径在肝癌发展中起重要作用 。.科学意义：.lncRNA XIST与GLS2和miR-424形成负向反馈回路促进肝癌的生长和侵袭，lncRNA XIST是肝癌的一个独立预后因素。